The present invention relates to electrophoresis generally and more particularly to apparatus for conducting an electrophoresis test therein.
A great deal of diagnostic procedures and laboratory research are carried out wherein DNA, RNA or proteins are separated according to their physical and chemical properties via electrophoresis. This process is widely used and has many applications. For example, it is used to analyze DNA molecules according to their resultant size after being digested by restriction enzymes. It is also used to analyze the products of a polymerase chain reaction (PCR).
Typically, electrophoresis separation is carried out in a separation medium, such as a gel of agarose or acylamide or a combination of the two. Usually, agarose gels are cast in open trays and form a slab whereas acrylamide gels are cast between two glass plates.
In order to effect the electrophoretic separation, two opposite ends of the gels are exposed to an electrically conducting buffer which is connected by electrodes, typically carbon or platinum, to an electric power source. Once the electrical power source is switched on, the electric field forces negatively charged molecules to move towards the anode and positively charged molecule to move towards the cathode. One characteristic of conventional electrophoresis is the use of large volumes of buffer having a relatively low salt concentration to maintain the required electric field.
DNA is negatively charged and therefore, in the agarose or acrylamide gels which provide sieving action, DNA molecules move towards the anode at a rate which depends on their size, wherein the smaller the molecules the faster they move.
In the electrophoretic separation of proteins, the proteins are often treated with an ionic detergent, such as sodium dodecylsulphate (SDS). The negatively charged dodecylsulphate anions interact with hydrophobic domains on the protein molecules, thus creating negatively charged protein/SDS complexes that undergoing electrophoresis separation similarly to DNA molecules.
Typically, it is desirable to visualize and to document the results of the electrophoretic separation test. In electrophoretic separation of DNA molecules, this has been done by immersing the gel slab after the electrophoretic separation has been completed in a solution of a fluorescent compound which emits visible light when exposed to a ultra violet (UV) light. A widely used compound in ethidium bromide.
Conventional electrophoretic separation systems are deficient in many respects, a few of which are listed below.
Prior art electrophoresis separation systems are a potential source of contamination to the working environment in which the tests are performed. The two major sources of contamination are ethidium bromide and PCR products. Ethidium bromide is a hazardous chemical due to its mutagenic activity and therefore, exposure to ethidium bromide may induce malignant tumors. PCR is an extremely sensitive method to the extent that a single molecule of DNA product from one PCR (out of the trillions of molecules being produced) may interfere with the subsequent PCR such that it will produce incorrect result.
Conventional electrophoresis is also deficient in other respects, one being that it is time consuming.
Various attempts have been made to solve the deficiencies of conventional electrophoresis. Most attempts have been addressed to overcome the deficiency of conventional electrophoresis systems with respect to the use of buffers therein.
U.S. Pat. No. 4,874,491 to Stalberg describes an electrophoresis system having a high concentration buffer containing gel.
U.S. Pat. No. 4,892,639 to Sarrine et al. describes an electrophoresis plate with improved buffer circulation.
U.S. Pat. No. 5,045,164 to Tansamrit et al. describes an electrophoresis plate having thickened ends as buffer reservoirs.
U.S. Pat. No. 5,209,831 to MacConnel describes a bufferless disposable cassette having open ends and conductive film electrodes.
U.S. Pat. Nos. 5,407,552 to Lebacq and 5,411,657 to Leka describe open electrophoresis devices requiring a buffer tank for operation.
It is therefore an object of the present invention to provide an improved apparatus for electrophoresis.
A major object of the present invention is to provide a closed cassette for electrophoresis which is substantially closed before, during and after an electrophoresis test conducted therein.
According to an aspect of the present invention the cassette is a disposable cassette.
The cassette of the present invention overcomes drawbacks associated with prior art electrophoresis cassettes, plates or slabs. Since the cassette is a closed one, its outer environment is not susceptible to contamination. Moreover, since it is ready to use, the preparation time required for preparing prior art cassettes is saved.
Another object of the present invention is to provide an electrophoresis system in which both the electrophoretic separation and the visualization of the results thereof are done while the cassette is in situ.
According to one aspect of the present invention, there is provided a substantially closed disposable cassette with openings for introducing a sample of molecules thereinto, the openings being preferably opened only just before the electrophoresis test.
According to another aspect of the present invention, the cassette includes all the chemical compounds required to drive the electrophoretic separation.
In accordance with yet another aspect of the present invention, when DNA, RNA and protein molecules are separated, the cassette includes the compounds required to stain the separated DNA, RNA and proteins, respectively.
According to yet another aspect of the invention, the volume of the ion source utilized for providing the ions required for the electrophoresis separation is smaller than the volume of the gel utilized as the electrophoresis separation matrix and preferably smaller than the volume of gel utilized for actual separation during an electrophoresis test.
According to a preferred embodiment of the present invention, the ions (cations and anions) required to drive the electrophoretic separation are provided by a cation exchange matrix and an anion exchange matrix, respectively.
According to another preferred embodiment of the present invention, the ions exchange matrix also provides the ions required to stain the separated molecules in order to enable visualization thereof when the cassette is illuminated with a UV light source in the case of DNA molecules separation and with a visible light source when protein molecules are being electorphoretically separated.
According to an alternative embodiment of the present invention the ions required to drive the electrophoresis separation are provided by a reservoir, preferably a breakable ampoule containing a buffer characterized by relatively high concentration of these ions.
One advantage of the cassette of the present invention is that it is disposable.
Another advantage of the cassette of the present invention is that the user is not exposed to any hazardous chemical constituent, such as ethidium bromide, as in prior art cassettes.
Yet another advantage of the cassette of the present invention is that PCR-DNA products are contained within the cassette and are disposed therewith so as to substantially reduce the contamination of the working environment in which the tests are performed.
There is thus provided, in accordance with a preferred embodiment of the present invention, an apparatus for conducting electrophoresis separation therein which includes a housing having at least bottom and side walls defining a chamber, wherein the chamber includes in contact therebetween a body of gel for carrying therein the electrophoresis separation, at least one ion source for providing ions for driving the electrophoresis, the at least one ion source having a volume smaller than the volume of the body of gel, and electrodes for connecting the chamber to an external electrical power source, thereby enabling to drive the electrophoresis separation.
There is also provided, in accordance with a preferred embodiment of the present invention a substantially closed cassette for conducting therein electrophoresis separation, the cassette being closed before, during and after electrophoresis separation, which includes a closed chamber which includes therein a body of gel for carrying therein the electrophoresis separation, at least one ion source for providing ions for driving the electrophoresis separation, and electrodes for connecting the cassette to an external electrical power source, thereby enabling to drive the electrophoresis separation.
According to a preferred embodiment, the volume of the at least one ion source is smaller than the volume of the body of gel utilized in the electrophoresis separation.
In a preferred embodiment, the at least one ion source includes a body of ion exchange matrix. Further, the body of ion exchange matrix includes a body of cation exchange matrix for providing the cations for driving the electrophoresis separation and a body of anion exchange matrix for providing the anions for driving the electrophoresis separation. Still further, the cation exchange matrix is disposed at one end of the body of separating gel and the body of anion exchange matrix is disposed on a second end of the separating gel.
In operation, the cation exchange matrix exchanges protons derived from electrolysis with the cations for driving the electrophoretic separation and the anion exchange matrix exchanges hydroxyl ions derived from the electrolysis with the anions for driving the electrophoretic separation.
According to a preferred embodiment of the present invention, the cation exchange matrix and the anion exchange matrix includes particles immersed in a support matrix. Preferably, the support matrix is formed of the gel as the body of gel for carrying the electrophoresis separation therein.
In accordance with yet a further embodiment of the present invention, the apparatus also include an additional body of gel of low gel strength disposed between the side wall of the chamber and the anion exchange matrix, the body of gel of low gel strength shrinking during the electrophoresis separation, thereby providing a volume in which gases produced at the vicinity of an anode of the chamber accumulates.
Further, according to a preferred embodiment of the present invention, the apparatus includes a buffer solution in contact with the body of separating gel, the at least one body of ion exchange matrix and the electrodes. Preferably, the buffer is a TAE buffer, thus the cation exchange matrix releases Tris cations and the anion exchange matrix releases acetate anions.
Additionally, according to a preferred embodiment of the present invention, the cation exchange matrix includes ethidium cations.
In accordance with an alternative embodiment of the present invention, the at least one ion source includes a closed reservoir having therein a buffer solution having higher concentrations than a concentration of a buffer solution of the body of gel for carrying therein the electrophoresis separation, the closed reservoir being opened just before the electrophoresis separation for providing the ions for driving the electrophoresis separation.
In a preferred embodiment, the closed reservoir is a breakable ampoule. Further, the breakable ampoule may be surrounded by a space, the space at least partially filled with the buffer solution in a concentration generally similar to that of the body of gel for carrying therein the electrophoresis separation. Preferably, the buffer is a TAE buffer. In addition, the buffer may also include ethidium cations.
The apparatus and cassette of the present invention are further characterized by any combination of the following features:
The chamber or the cover may include at least one opening therein for introducing at least one test sample into the body of gel. Preferably, the at least one opening is closed by a comb prior to the electrophoresis separation.
The chamber and/or the cover may be transparent to ultra violet (UV) radiation.
The chamber or cover may also include at least one vent hole which is closed prior to the electrophoresis test and is being opened just before the electrophoresis test.
Further, according to a preferred embodiment of the present invention, the electrodes include a conductive material capable of adsorbing at least part or at least one of the gases produced during the electrophoresis separation. Preferably, the at least one electrode capable of adsorbing is substantially formed from a material selected from the group consisting of aluminum and palladium.
Additionally, the gases include oxygen created at the vicinity of the anode during the electrophoresis separation and reacting with the aluminum. Alternatively, the gases include hydrogen created at the vicinity of the cathode during the electrophoresis separation and wherein the hydrogen is adsorbed by the palladium.
In an alternative embodiment, the at least one electrodes includes a strip of conductive material. Preferably, the strip of conductive material is mounted on a ramp, the ramp being inclined at an angle relative to the bottom wall, whereby gases produced at the vicinity of the strip during the electrophoresis separation are being directed to an empty volume receiving the gases.
Finally, the apparatus or cassette may also include at least one empty volume for accumulating gases produced during the electrophoresis test.
There is also provided, in accordance to a preferred embodiment of the present invention a system for conducting electrophoresis separation which includes an electrical power source, a substantially closed disposable cassette substantially closed before, during and after electrophoresis separation therein, preferably, but not necessarily, the apparatus or cassette of the present invention, and a support for supporting the substantially closed cassette and for connecting the electrical power source to the conductive elements of the cassette.
Further, the system may also include a UV light source and wherein the cassette is transparent to UV light, and wherein the cassette also includes a UV sensitive material capable of interacting with the molecules undergoing electrophoresis separation and of emitting light, thereby enabling to conduct the electrophoresis separation and to visualize it while the cassette is in situ. In a preferred embodiment, the UV sensitive material is ethidium bromide.
Still further, the system may also include camera means for documenting the results of the electrophoresis separation. The system may also include a computer which includes at least one image analysis application for analyzing the results of the electrophoresis separation.
Additionally, the system may include a cooling system for cooling the cassette during the electrophoresis test.
There is also provided, in accordance with a preferred embodiment of the present invention, an electrophoresis method which includes the steps of introducing at least one test sample into a body of gel, applying an electrical field to the body of gel and driving an electrophoresis separation by providing ions required for driving the electrophoresis separation by at least one ion source having a volume smaller than the volume of the gel.
Further, there is also provided, in accordance with a preferred embodiment of the present invention, a method for producing a substantially closed cassette for conducting electrophoresis separation therein which includes the steps of providing a housing having bottom and side walls defining an open chamber, assembling within the chamber in contact therebetween a body of gel for carrying therein the electrophoresis separation, at least one ion source for providing ions for driving the electrophoresis separation, the at least one ion source having a volume smaller than that of the body of gel and electrodes for connecting the chamber to an external electrical power source, and closing the open housing with a cover, thereby forming a substantially closed cassette capable of carrying the electrophoresis separation therein.
Still further, there is also provided, according to yet another preferred embodiment of the present invention apparatus for conducting electrophoresis separation therein which includes a chamber having therein a body of separating gel for carrying therein the electrophoresis separation and electrodes for connecting the chamber to an external electrical power source, thereby driving the electrophoresis separation, at least one of the electrodes also providing ions for driving the electrophoresis separation.
Further, according to a preferred embodiment, the electrophoretic separation is substantially free from water electrolysis and the pH is generally constant throughout the body of separating gel.
Still further, the chamber may also include a dye source for providing a dye, the dye enabling visualization of the electrophoresis separation.
In accordance with one preferred embodiment, the electrode providing the ions is the anode. Further, during the electrophoresis separation an electrochemical reaction wherein metal atoms of the anode loose electrons and enter solution as cations occurs preferentially to electrolysis of water molecules.
In a preferred embodiment, the anode is selected from the group consisting of lead, silver and copper.
According to yet another preferred embodiment, the anode is in contact with a body of a cation exchange matrix for binding the ions provided by the anode thereby releasing other ions for driving the electrophoresis separation.
In an alternative embodiment, the cathode is in contact with a salt suspended in a support matrix, the salt is insoluble in water. During electrophoresis separation an electrochemical reaction wherein cations of the salt receive electrons from the cathode occurs preferentially to electrolysis of water molecules.
In a preferred embodiment, the cathode is selected from the group consisting of lead and silver and copper and the salt is selected from the group consisting of lead carbonate and silver chloride.
In another preferred embodiment, the anode is in contact with a salt suspended in a support matrix and the cathode end is in contact with a body of cation exchange matrix.
In yet another preferred embodiment, the chamber includes a body of stacking gel, the body of stacking gel being in contact with the body of separating gel.